The Endocytic Recycling Protein EHD2 Interacts with Myoferlin to Regulate Myoblast Fusion
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m802306200
ISSN1083-351X
AutoresKatherine R. Doherty, Alexis R. Demonbreun, Gregory Q. Wallace, Andrew Cave, Avery D. Posey, Konstantina Heretis, Peter Pytel, Elizabeth M. McNally,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoSkeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion. Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion. Muscle development and regeneration rely on the fusion of singly nucleated myoblasts to initiate the formation or augment the growth of multinucleated myotubes. Myoblast fusion involves multiple steps including cell recognition, adhesion, and membrane coalescence. Genetic analyses of Drosophila mutants that fail to form normal muscles during development have been instrumental in elucidating not only the critical steps of myoblast fusion but also proteins that mediate recognition, adhesion, and the cytoskeletal elements that mediate fusion (1Chen E.H. Olson E.N. Trends Cell Biol. 2004; 14: 452-460Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In mammalian cells additional proteins that regulate myoblast fusion have been characterized, as well as many proteins implicated in myoblast differentiation (2Horsley V. Pavlath G.K. Cells Tissues Organs. 2004; 176: 67-78Crossref PubMed Scopus (193) Google Scholar, 3Krauss R.S. Cole F. Gaio U. Takaesu G. Zhang W. Kang J.S. J. Cell Sci. 2005; 118: 2355-2362Crossref PubMed Scopus (133) Google Scholar, 4Taylor M.V. Curr. Biol. 2003; 13: 964-966Abstract Full Text Full Text PDF PubMed Google Scholar). In both vertebrate and invertebrate systems, ultrastructural analysis of fusing myoblasts reveal the presence of vesicles accumulating at the sites of membrane fusion (5Doberstein S.K. Fetter R.D. Mehta A.Y. Goodman C.S. J. Cell Biol. 1997; 136: 1249-1261Crossref PubMed Scopus (186) Google Scholar, 6Kalderon N. Gilula N.B. J. Cell Biol. 1979; 81: 411-425Crossref PubMed Scopus (116) Google Scholar). Because vesicle fusion is a common feature of most cells, it is possible that aspects of the myoblast fusion machinery utilize pathways more commonly used by other cell types for intracellular trafficking. Based on their involvement in vesicular fusion, the ferlin proteins are candidates for mediating the membrane coalescence stage of myoblast fusion. In Caenorhabditis elegans, mutations in the gene encoding the prototypical ferlin protein, fer-1, produce defective membrane fusions in developing spermatozoa that lead to fertility defects (7Achanzar W.E. Ward S. J. Cell Sci. 1997; 110: 1073-1081Crossref PubMed Google Scholar, 8Washington N.L. Ward S. J. Cell Sci. 2006; 119: 2552-2562Crossref PubMed Scopus (110) Google Scholar). Myoferlin is a member of the ferlin family and shares homology with fer-1; both proteins each contain multiple C2 domains in the long cytoplasmic portion followed by a carboxyl-terminal transmembrane domain. C2 domains consist of ∼130 amino acids that adopt conformation independent of neighboring sequences. Crystallographic studies of C2 domains support a structure with multiple aligned β strands and a calcium binding motif formed at one end from the intervening loops (9Benes C.H. Wu N. Elia A.E. Dharia T. Cantley L.C. Soltoff S.P. Cell. 2005; 121: 271-280Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 10Ochoa W.F. Torrecillas A. Fita I. Verdaguer N. Corbalan-Garcia S. Gomez-Fernandez J.C. Biochemistry. 2003; 42: 8774-8779Crossref PubMed Scopus (72) Google Scholar, 11Pappa H. Murray-Rust J. Dekker L.V. Parker P.J. McDonald N.Q. Structure. 1998; 6: 885-894Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). There are ∼100 different C2 domain-containing proteins, and many of these show membrane association. Most C2 domain-containing proteins have one or two domains where one may participate in binding phospholipids, whereas the second may participate in protein-protein interactions. The C2 domains in myoferlin are highly related to those in the synaptotagmins; synaptotagmins participate in the rapid exocytosis that occurs at nerve terminals. Synaptotagmins have two C2 domains, one of which binds phospholipids in response to calcium (12Tang J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Models for synaptotagmin function suggest that its C2 domains can insert directly within a lipid bilayer and participate in the fusion of two independent membranes (13Hui E. Bai J. Chapman E.R. Biophys. J. 2006; 91: 1767-1777Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Myoferlin and members of the ferlin family are unique because of their multi-C2 domain nature. The myoferlin amino-terminal C2 domain, C2A, directly binds negatively charged phospholipids in response to calcium (14Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). Myoferlin is abundantly expressed in myoblasts that are preparing to undergo fusion. In the elongated "prefusion" myoblast, myoferlin is found within vesicular structures in the cytoplasm and concentrated near the plasma membrane. In fusing myoblasts, myoferlin is concentrated at the sites of fusion (14Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). Myoferlin null myoblasts fuse poorly in culture leading to smaller myotubes, and in vivo, the loss of myoferlin leads to reduced muscle mass with the loss of large diameter myofibers (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). The Eps15 homology domain (EH domain), 3The abbreviations used are: EH, Eps15 homology; EHD, EH domain; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter. is a conserved domain important for protein interactions during vesicular trafficking (16Mayer B.J. Curr. Biol. 1999; 9: 70-73Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Santolini E. Salcini A.E. Kay B.K. Yamabhai M. Di Fiore P.P. Exp. Cell Res. 1999; 253: 186-209Crossref PubMed Scopus (115) Google Scholar). The mammalian genome encodes four carboxyl-terminal EH domain-containing proteins, EHD1–4 (18Confalonieri S. Di Fiore P.P. FEBS Lett. 2002; 513: 24-29Crossref PubMed Scopus (83) Google Scholar, 19Naslavsky N. Caplan S. J. Cell Sci. 2005; 118: 4093-4101Crossref PubMed Scopus (89) Google Scholar, 20Pohl U. Smith J.S. Tachibana I. Ueki K. Lee H.K. Ramaswamy S. Wu Q. Mohrenweiser H.W. Jenkins R.B. Louis D.N. Genomics. 2000; 63: 255-262Crossref PubMed Scopus (83) Google Scholar). These carboxyl-terminal EHD proteins are characterized by an amino-terminal nucleotide binding domain followed by a central coiled-coil domain (21Lee D.W. Zhao X. Scarselletta S. Schweinsberg P.J. Eisenberg E. Grant B.D. Greene L.E. J. Biol. Chem. 2005; 280: 17213-17220Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Functionally, these carboxyl-terminal EHD proteins have been linked to endosomal trafficking including the recycling of cell surface receptors back to the plasma membrane (19Naslavsky N. Caplan S. J. Cell Sci. 2005; 118: 4093-4101Crossref PubMed Scopus (89) Google Scholar, 22Rapaport D. Auerbach W. Naslavsky N. Pasmanik-Chor M. Galperin E. Fein A. Caplan S. Joyner A.L. Horowitz M. Traffic. 2006; 7: 52-60Crossref PubMed Scopus (64) Google Scholar, 23Daumke O. Lundmark R. Vallis Y. Martens S. Butler P.J. McMahon H.T. Nature. 2007; 449: 923-927Crossref PubMed Scopus (246) Google Scholar). C. elegans and Drosophila each contain a single EHD gene. In the worm, mutations in the presumed nucleotide binding domain of EHD, also known as RME-1, lead to delay of recycling membrane components after endocytosis (24Grant B. Zhang Y. Paupard M.C. Lin S.X. Hall D.H. Hirsh D. Nat. Cell Biol. 2001; 3: 573-579Crossref PubMed Scopus (218) Google Scholar). This role is conserved in mammals (25Lin S.X. Grant B. Hirsh D. Maxfield F.R. Nat. Cell Biol. 2001; 3: 567-572Crossref PubMed Scopus (213) Google Scholar). Mice engineered to lack EHD1 similarly display reduced endocytic recycling (22Rapaport D. Auerbach W. Naslavsky N. Pasmanik-Chor M. Galperin E. Fein A. Caplan S. Joyner A.L. Horowitz M. Traffic. 2006; 7: 52-60Crossref PubMed Scopus (64) Google Scholar). EHD-binding proteins have been described that regulate interactions with the cytoskeleton, the small GTPases, and several cell surface receptors that may be carried as cargo as they are returned to the plasma membrane (26Braun A. Pinyol R. Dahlhaus R. Koch D. Fonarev P. Grant B.D. Kessels M.M. Qualmann B. Mol. Biol. Cell. 2005; 16: 3642-3658Crossref PubMed Scopus (136) Google Scholar, 27Naslavsky N. Boehm M. Backlund Jr., P.S. Caplan S. Mol. Biol. Cell. 2004; 15: 2410-2422Crossref PubMed Scopus (114) Google Scholar, 28Guilherme A. Soriano N.A. Bose S. Holik J. Bose A. Pomerleau D.P. Furcinitti P. Leszyk J. Corvera S. Czech M.P. J. Biol. Chem. 2004; 279: 10593-10605Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 29Picciano J.A. Ameen N. Grant B.D. Bradbury N.A. Am. J. Physiol. Cell Physiol. 2003; 285: 1009-1018Crossref PubMed Scopus (78) Google Scholar). To understand better the components that direct myoblast fusion, we evaluated myoferlin interacting proteins in myoblasts undergoing fusion. Using immunoprecipitation and mass spectrometry, we identified EHD2 as a myoferlin-associated protein. Myoferlin contains the amino acid sequence asparagine-proline-phenylalanine (NPF), a known EHD binding motif, within its second C2 domain, and it is this region of myoferlin that binds directly to EHD2. EHD2 protein expression was significantly reduced in myoferlin null myoblasts. Moreover, myoferlin null myoblasts display delayed endocytic recycling accompanied by intracellular aggregation of labeled transferrin. Expression of mutant EHD2 protein in myoblasts leads to sequestration of myoferlin and an inhibition of myoblast fusion. This work demonstrates that impaired endocytic recycling is associated with defective myoblast fusion. Culture of C2C12 Cells and Primary Myoblasts—C2C12 cells were obtained from ATCC (catalog #CRL-1772). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin in 7% CO2. Primary myoblasts were isolated from neonatal wild type and myoferlin null pups and grown and maintained as described (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). All tissue culture media and sera used were from Invitrogen. Immunoprecipitation—C2C12 cells or primary myoblasts were grown to confluency (5 days) in growth media. Cells were lysed in coimmunoprecipitation buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 0.15% CHAPS with Complete Mini Protease Inhibitor mixture, Roche Applied Science). Cellular debris was removed, and the protein concentration of the supernatant was determined using Bio-Rad protein assay. Two hundred μl of protein G-Sepharose beads (Amersham Biosciences) was blocked for 1 h with 20 mg/ml bovine serum albumin in coimmunoprecipitation buffer, preincubated for 30 min with anti-myoferlin antibody, MYOF3 (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar), then incubated overnight with 200 μg of protein. Beads were washed 4 times in coimmunoprecipitation buffer and boiled 10 min in loading buffer (50 mm Tris, pH 6.8, 100 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), and the supernatant was loaded on a 4–20% acrylamide gel. Gels were either silver-stained (Bio-Rad Silver Stain Plus) or stained with Coomassie or transferred to polyvinylidene difluoride Immobilon-P membrane (Millipore). Identification of Interacting Proteins—The band of interest was excised from a Coomassie-stained acrylamide gel. Five bands of the same molecular weight were combined and subjected to MALDI-TOF mass spectrometry at the Proteomics Core Facility at University of Chicago. Using the Mascot search engine, full-length murine proteins corresponding to the peptides generated by the mass spectrometry were identified in the non-repetitive NCBI data base, and statistical significance of the matches was determined. Immunoblotting—Proteins were transferred to polyvinylidene difluoride membrane, stained with Ponceau as a loading control, and immunoblotted with polyclonal anti-myoferlin (MYOF3, 1:2000) (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar), polyclonal goat anti-EHD2 (1:10–25,000, Abcam), or anti-Xpress (1:5000, Invitrogen). Secondary antibodies, goat anti-rabbit, goat anti-mouse, and donkey anti-goat conjugated to horseradish peroxidase (Jackson ImmunoResearch) were used at a dilution of 1:5000. Blocking and antibody incubations were done in 5% milk in 1× Tris-buffered saline with 0.1% Tween 20 for all antibodies except anti-EHD2 and donkey anti-goat conjugated to horseradish peroxidase. These were incubated in 1× Tris-buffered saline containing 4% donkey serum and 0.1% Tween 20. ECL-Plus chemiluminescence (Amersham Biosciences) and Kodak Biomax MS film or a Amersham Biosciences PhosphorImager was used for detection. In Vitro Binding Experiments—Human myoferlin C2 domains were ligated into pGEX4T-1 as previously described (14Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Mutant versions of myoferlin C2B were generated by site-directed mutagenesis using Pfu polymerase and the following sets of primers: AAG AGA GGA AAC AGC CCT TTG TTT GAT GAG TTG TT and AAC AAC TCA AAC AAA GGG CTG TTT CCT CTC TCT T (NPF to dysferlin C2B sequence SPL) and AAG AGA GGA AAC TGC CCT TTT TTT GAT GAG TTG TT and AAC AAC TCA AAA AAA GGG CTG TTT CCT CTC TCT T (NPF to fer1L4 C2B sequence CPF) as described (14Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Cultures of BL21 cells (Invitrogen) expressing glutathione S-transferase-myoferlin C2 domain fusion proteins were induced for 1 h with isopropyl 1-thio-β-d-galactopyranoside at a final concentration of 85 μg/μl. Cells were resuspended in 10 mm Tris, pH 7.5, and added to SDS loading buffer. After boiling, 10 μl of lysed cells was loaded on 10% acrylamide gel. For each experiment two gels were used; one was transferred to polyvinylidene difluoride membrane used for gel overlay, and the other was stained with GelCode (Bio-Rad). A clone corresponding to the full-length murine cDNA of EHD2 (NCBI accession number AK046566) was obtained from RIKEN (clone ID B430101D12). The cDNA was amplified with the primers EHD2 (forward, ATG TTC AGC TGG CTG AAG AGG GG; reverse, TCA TTC AGC AGA GCC CTT CTG TC) and cloned into pCR T7/NT-TOPO (Invitrogen). 35S-Labeled in vitro transcribed/translated EHD2 was generated using [35S]methionine with the Promega TnT Coupled Reticulocyte Lysate system. The product was separated on a G-25 Sephadex column (Roche Applied Science). The membrane was blocked for 2 h at 4 °C in blocking buffer (0.1% gelatin, 5% bovine serum albumin, 0.1% Tween in 1× PBS, pH .5) then incubated overnight at 4 °C in overlay buffer (150 mm NaCl, 20 mm HEPES, 2 mm MgCl2, 5% bovine serum albumin, pH 7.5) containing the radioactive EHD2 as described (30Chan Y. Kunkel L.M. FEBS Lett. 1997; 410: 153-159Crossref PubMed Scopus (23) Google Scholar). The membrane was washed 4 × 20 min at room temperature in overlay buffer and exposed to film and/or imaged on a PhosphorImager. Immunostaining and Microscopy—Primary myoblasts were grown on gelatin-coated glass coverslips, fixed in 4% paraformaldehyde for 10 min on ice, rinsed with PBS, then fixed 2 min on methanol on ice. C2C12 cells were fixed in 4% paraformaldehyde 10 min on ice followed by 10 min in 0.3% Triton-X100 in 1× PBS on ice. Polyclonal MYOF2 serum (31Davis D.B. Delmonte A.J. Ly C.T. McNally E.M. Hum. Mol. Genet. 2000; 9: 217-226Crossref PubMed Scopus (142) Google Scholar) was used at 1:200, goat polyclonal anti-EHD2 (Abcam, ab23935) was used at 1:500, and monoclonal anti-Xpress (Invitrogen) was used at 1:500. Donkey anti-rabbit conjugated to Alexa488, donkey anti-goat conjugated to Alexa 594, goat anti-rabbit conjugated to Alexa 594, and goat anti-mouse conjugated to Alexa488 were obtained from Molecular Probes and used at 1:2000. Blocking and antibody incubations were done in 1× PBS, 4% donkey serum, 0.1% Triton X-100 with donkey anti-goat and donkey anti-rabbit secondary antibodies. With goat anti-mouse and goat anti-rabbit secondary antibody incubations, blocking and antibody incubations were done in 1× PBS, 5% fetal bovine serum, 0.1% Triton X-100. Coverslips were mounted using Pro-Long Gold with 4′,6-diamidino-2-phenylindole. Images were captured using either a Zeiss Axiophot microscope and Axiovision software (Carl Zeiss) or a Leica SP2 scanning laser confocal microscope and LCS Leica Confocal Software. Transferrin Internalization Assay—Myoblasts were grown on sodium hydroxide-treated glass coverslips. Cells were incubated in Dulbecco's modified Eagle's medium, 0.1% bovine serum albumin, 20 mm HEPES, pH 7.2. Cells were incubated with Alexa488-conjugated transferrin (25 μg/ml, Molecular Probes) for 60 min followed by chase with 50 mm deferoxamine, 250 μg/ml holotransferrin in 20% fetal bovine serum, 20 mm HEPES, pH 7.2. Cells were fixed in 4% paraformaldehyde and mounted for visualization. Myoblast cultures analyzed by flow cytometry were prepared as above but were trypsinized then fixed in 4% paraformaldehyde. Cells were washed and resuspended in PBS for analysis in a FACS Canto (BD Biosciences). After gating to remove dead cells, aggregated cells, and debris, a minimum of 20,000 events was scored per time point per culture. Cultures were derived from three independent animals per genotype. Fluorescent gates were set to include 97% of wild type cells at time 0 after transferrin removal. The percentage of fluorescent cells was determined on subsequent time points by the number of cells remaining in this gate. Median fluorescence values were determined using a histogram of Alexa488 fluorescence. FACS data were analyzed using FlowJo software (Tree Star, Inc.). Expression of Wild Type and G65R EHD2—The full-length EHD2 cDNA was amplified with the primers EHD2 (forward, GGT ACC CAT GTT CAG CTG GCT GAA GAA GGG C; reverse, GCG GCC GCT CAT TCA GCA GAG CCT TCT GTC GT) and ligated into the KpnI and NotI sites of pcDNA3.1 His B (Invitrogen), which contains an amino-terminal Xpress epitope tag and a cytomegalovirus promoter. The G65R mutation was generated by site-directed mutagenesis using the primers EHD2 G65R (forward, GTG CTG GTG GCC CGC CAG TAT AGC ACC GGC; reverse, G65R GCC GGT GCT ATA CTG GCG GGC CAC CAG CAC) and Pfu polymerase as described (14Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. J. Biol. Chem. 2002; 277: 22883-22888Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). C2C12 cells were plated at 50,000 cells per well on glass coverslips in 6-well plates. The following day each well was transfected with 5 mg of DNA using Lipofectamine Plus and Opti-MEM (Invitrogen). Twenty four hours after transfection, cells were differentiated or fixed, stained, and imaged as described above. Identification of EHD2 as a Myoferlin Interacting Protein—Because myoferlin is abundantly expressed in prefusion myoblasts, C2C12 cells were grown to confluence in 10% fetal bovine serum to enrich for myoblasts in the early phases of fusion. As shown in Fig. 1, the polyclonal anti-myoferlin antibody, MYOF3, immunoprecipitated myoferlin as well as a protein of ∼65 kDa (arrow in Fig. 1B). After determining that this band appeared reproducibly, the results of repeated experiments were combined and analyzed by MALDI-TOF mass spectrometry. The Mascot search engine (32Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar) was used to identify full-length proteins in the NCBI data base corresponding to the peptides generated by mass spectrometry analysis and to determine which matches were statistically significant. A probability-based Mowse score was determined, and a significance score was set at p < 0.5 for this analysis. From this approach, fourteen full-length proteins contained a sufficiently high match to peptides identified by mass spectrometry. Of these 14, 13 were likely contaminants, as they encoded keratin-related intermediate filament proteins (8 of 14), α globin, the β-2 chain of hemoglobin, complement C3, and two RNA-binding proteins. Two peptides in this analysis corresponded to EHD2 (Eps15 homology domain 2), and the position of these sequences along the EHD2 schematic is indicated in Fig. 1A. We selected this protein for further analysis because EHD2 is a protein of the expected size that localizes in the cytoplasm and the membrane similar to myoferlin. Eps15 homology (EH) domains are found in more than 50 eukaryotic proteins, most of which are involved in regulating membrane traffic and events such as receptor internalization, vesicle transport, and actin polymerization (33Polo, S., Confalonieri, S., Salcini, A. E., and Di Fiore, P. P. (2003) Sci. STKE, re17Google Scholar). EHD2 is a member of a subclass of EH domain-containing proteins differentiated by the presence of a carboxyl-terminal EH domain. EHD2 is predicted to be 61 kDa, very close to the observed size of the protein co-immunoprecipitated by the anti-myoferlin antibody. We immunoblotted cell extracts from the myoblast C2C12 line demonstrating that EHD2 consistently migrated slower than the 62-kDa marker at ∼65 kDa (arrow in Fig. 1C). In addition, immunoblotting with an anti-EHD2 antibody confirmed that EHD2 was immunoprecipitated along with myoferlin in the presence of anti-myoferlin antibodies (Fig. 1D). The antibody to EHD2 was unable to immunoprecipitate EHD2 and, thus, could not be used to determine whether EHD2 antibodies could immunoprecipitate myoferlin (data not shown). EHD2 and Myoferlin Interact Directly—The Eps15 EH domain is composed of ∼100 amino acids, and EH domains are 50–60% conserved among proteins (34Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9530-9534Crossref PubMed Scopus (136) Google Scholar). NMR spectroscopy has shown that EH domains contain two EF-hands linked by an anti-parallel sheet (18Confalonieri S. Di Fiore P.P. FEBS Lett. 2002; 513: 24-29Crossref PubMed Scopus (83) Google Scholar). The EH domain recognizes and binds to the motif asparagine-proline-phenylalanine (NPF) (35Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Di Fiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (287) Google Scholar). This sequence was found in both human and murine myoferlin at amino acids 238–240. When engaged, the NPF motif is predicted to project into a conserved hydrophobic pocket in the EH domain where a conserved tryptophan interacts with the asparagine (36de Beer T. Carter R.E. Lobel-Rice K.E. Sorkin A. Overduin M. Science. 1998; 281: 1357-1360Crossref PubMed Scopus (111) Google Scholar, 37de Beer T. Hoofnagle A.N. Enmon J.L. Bowers R.C. Yamabhai M. Kay B.K. Overduin M. Nat. Struct. Biol. 2000; 7: 1018-1022Crossref PubMed Scopus (90) Google Scholar). The C2B domain of myoferlin shows significant homology to the C2A domain of synaptotagmin I (Fig. 2A). Using the known structure of synaptotagmin, we modeled the myoferlin C2B domain. With this configuration, the NPF motif is located at the beginning of the fourth β strand, protruding from the surface of the domain and accessible for protein-protein interactions (Fig. 2B). Because C2 domains have been previously shown to fold independent of their surrounding sequences (38Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar), we assessed each C2 domain of myoferlin individually. We demonstrated a specific and direct interaction between the NPF amino acids in myoferlin C2B and EHD2 (Fig. 2C). EHD2 bound directly to myoferlin C2B but did not bind detectably to the other C2 domains in myoferlin including C2A, C2C, C2D, C2E, or C2F. Consistent with this lack of binding, none of these domains possesses the NPF motif. Two different mutants of the NPF motif were generated to document the specificity of the EHD2-myoferlin interaction. In both mutants we left intact the central proline residue that forms the core of the NPF motif to avoid disrupting the entire C2 domain. The first mutant tested (SPL) represents the residues found in the related protein dysferlin, and the second mutant (CPF) represents the sequences found in fer1l4, another ferlin sequence. This experiment was performed in triplicate and quantified to show that EHD2 binding to both mutants was reduced by 4-fold (Fig. 2D). This binding was not affected by the presence of calcium, and this is consistent with the lack of calcium binding of C2B (data not shown). EHD2 Is Reduced in Myoferlin Null Myoblasts—We found that EHD2 was expressed in myoblasts isolated from muscle. As shown in Fig. 3, EHD2 was expressed in undifferentiated myoblasts enriched near the plasma membrane in punctate structures. When myoblasts were co-stained with the anti-myoferlin antibody MYOF2 and anti-EHD2, overlap in the merged images was seen (Fig. 3A). Confocal imaging (Fig. 3A) showed myoferlin and EHD2 immunostaining at sites enriched along the plasma membrane, consistent with colocalization and a potential role in vesicle trafficking. Myoferlin null mice were previously characterized and have a phenotype of reduced muscle mass and smaller diameter myofibers (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). When cultured, myoferlin null myoblasts do not fuse efficiently and generate smaller myotubes (15Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar). Myoferlin null myoblasts showed a marked reduction in EHD2 compared with wild type control myoblasts (Fig. 3B) and was confirmed by immunoblotting undifferentiated primary myoblasts from control and myoferlin null mice (Fig. 3C). Delayed Endocytic Recycling in Myoferlin Null Myoblasts—Transferrin is a small molecule that is rapidly internalized into cells via the transferrin receptor. Once internalized, transferrin is recycled to the plasma membrane where it can be released to bind iron. Fluorescently labeled transferrin is used commonly to image and measure endocytic recycling. Myoferlin null and wild type control myoblasts were isolated and incubated with fluorophore-labeled transferrin. M
Referência(s)